Protein S protects the nervous system from injury

ABSTRACT

Protein S is a significant neuroprotectant when administered after focal ischemic stroke and prevents hypoxic/re-oxygenation injury. Purified human plasma-derived or recombinant protein S improves motor neurological function after stroke, and reduced brain infarction and edema. Protein S also enhances post-ischemic reperfusion and reduced brain fibrin and neutrophil deposition. Cortical neurons are protected from hypoxia/re-oxygenation-induced apoptosis. Thus, protein S and variants thereof are prototypes of a class of agents for preventing injury of the nervous system. In particular, a disease or other pathological condition (e.g., stroke) may be treated with such agents having one or more protein S activities (e.g., anti-thrombotic and anti-inflammatory activities, direct cellular neuronal protective effects) although the latter activities are not be required.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of International PatentAppln. No. PCT/US2003/030638, filed Sep. 30, 2003; which claims thebenefit of provisional Appln. No. 60/414,333, filed Sep. 30, 2002; whichis incorporated by reference herein.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has certain rights in this invention as provided byNIH grants HL63290 and HL21544 from the Department of Health and HumanServices.

BACKGROUND OF THE INVENTION

This invention relates to the use of protein S and/or variants thereofas neuroprotective agents for treating brain disorders and otherpathological conditions. The ability of protein S and variants thereofto act as cell survival factors on cells of the nervous systems aredemonstrated.

Benzakour and Kanthou (Blood 95:2008-2014, 2000) showed that protein Sis produced by smooth muscle cells derived from abdominal arteries. Theysuggested that protein S may be an important autocrine factor in thepathophysiology of the vasculature by acting as a mitogen for thesecells. In more recent work, they induced apoptosis of abdominal vessel,smooth muscle cells using sodium nitroprusside or hydrogen peroxide.Pre-treatment with protein S reduced apoptosis and cell death (Kanthou &Benzakour in Angio-genesis: From the Molecular to IntegrativePharmacology, pp. 155-166, 2000).

These studies did not address the role of protein S in the nervoussystem or, more specifically, its effects on brain endothelial cells.

The present invention addresses the need for neuroprotectivecompositions and methods for their use to treat diseases associated witha variety of types of nervous system damage, thrombosis, andinflammation. Because injury usually occurs after a triggering event,treatment may be initiated after such an event.

Therefore, it is an objective of the invention to show how to useprotein S and variants thereof as neuroprotective agents. A long-feltneed for new therapeutic and prophylactic compositions is addressedthereby. Also provided are compositions that have been formulated todeliver protein S or variants thereof to the central nervous system andprocesses for using and making the aforementioned products. Furtherobjectives and advantages of the invention are described below.

SUMMARY OF THE INVENTION

The present invention is directed to improved protection of cells of thenervous system. An effective amount of protein S or at least one variantthereof may be used. It may or may not have one or more optionalactivities: for example, inhibition of any combination of cellularstress, apoptosis, injury, or cell death; prevention of cell injury ortissue damage caused by ischemia, hypoxia, re-oxygenation, reperfusion,or the like; and anti-thrombotic and/or anti-inflammatory activity.

The subject in need of treatment may be at risk for or already affectedby the disease or other pathological condition. Treatment may beinitiated before and/or after diagnosis. An indication that treatment iseffective may be increased function or improved neurological outcomeincluding improved motor neurological performance, improved performanceon psychiatric tests, improved level of cognitive performance; decreasedbrain damage due to head injury, ischemic injury, infarction, edema, ora combination thereof; decreased injury of the nervous system; orincreased cerebral blood flow. Increase or decrease may be determined bycomparison to treatment without protein S or variant thereof, or to theexpected effects of untreated disease or another: pathologicalcondition. Other advantages and improvements are discussed below orwould be apparent from the disclosure herein.

Therefore, the invention provides a treatment for therapy or prophylaxisand the products used therein. Pharmaceutical compositions may bemanufactured and assessed in accordance therewith. Further aspects ofthe invention will be apparent to persons skilled in the art from thefollowing detailed description and claims, and generalizations thereto.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1A-1B show cerebral blood flow (CBF) during middle cerebral artery(MCA) occlusion/reperfusion in control (FIG. 1A) and protein S-treatedmice (FIG. 1B). Vehicle or protein S (2 mg/kg) was given 10 min afterinitiation of the MCA occlusion. CBF values (mean ±SD) in the ischemic(open symbols) and non-ischemic hemisphere (closed symbols) in sixcontrols and six protein S-treated mice were measured. *p<0.05 betweenthe two groups.

FIGS. 2A-2D show brain injury in control and protein S-treated mice.FIGS. 2A to 2C: The volumes of brain injury, infarction and edema (mean±SE) from control mice (n=6); mice treated with human plasma-derivedprotein S at 0.2 mg/kg (n=5), 0.5 mg/kg (n=6) or 2 mg/kg (n=6); and micetreated with recombinant protein S^(REC) at 2 mg/kg (n=4); *p<0.01,**p<0.05, #p=0.059. FIG. 2D: Infarct area in the seven coronal sectionsfrom the brains of control mice and mice treated with 0.5 mg/kg proteinS (mean ±SE); * p<0.05. Vehicle or protein S was given 10 min afterinitiation of MCA occlusion.

FIGS. 3A-3B show the incidence and topography of the infarction at thelevel of the optic chiasm in control mice (FIG. 3A) and mice receivingplasma-derived protein S (FIG. 3B). Key for the incidence is given inFIG. 3A. Vehicle (n=6) or protein S (2 mg/kg, n=6) was given 10 minafter initiation of MCA occlusion.

FIGS. 4A-4C show deposition of fibrin at the level of optic chiasm andCD11b-positive leukocytes in the ischemic and non-ischemic hemispheresin control mice and protein S-treated (2 mg/kg) mice. FIG. 4A: Signal onWestern blot for fibrin from the standard curve was linear between 0.15and 3 μg of fibrin β-chain/0.1 ml; 3 μg/0.1 ml was arbitrarily set as 1unit. FIG. 4B: Western blot analysis of 10 mg brain homogenate (mean±SE)in control (n=3) and protein S-treated mice (n=3). FIG. 4C:CD11b-positive leukocytes (mean±SE) from six controls (open bars) andsix protein S-treated mice (closed bars). *p<0.05.

FIGS. 5A-5D show neuroprotective effects of protein S in cultured mousecortical neurons subjected for 12 hr to hypoxia/aglycemia followed by 12hr re-oxygenation. FIG. 5A: TUNEL-positive neurons (upper) and neuronsshowing chromatin condensation and/or nuclear fragmentation by Hoechststaining (lower) under normoxic conditions (left),hypoxia/re-oxygenation (middle) and hypoxia/re-oxygenation with proteinS (500 nM, right). FIG. 5B: TUNEL-positive neurons in the absence orpresence of protein S or recombinant protein S^(REC) corrected for basalvalues of apoptosis. FIG. 5C: Time-course for anti-apoptotic effect ofprotein S. FIG. 5D: Dose-response for neuroprotective effect of proteinS. Hypoxia/re-oxygenation in the absence (open squares) or presence ofincreasing concentrations of protein S (solid squares). Mean±SE, from 3to 5 cultures. *p<0.05 and **p<0.01.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Protein S is a physiologic anti-thrombotic agent that inhibitsprothrom-binase complex activity on endothelial cells and platelets byinhibiting coagulation factors Va and Xa. Protein S is also a cofactorfor activated protein C, a serine protease that inactivates coagulationfactors Va and VIIIa (1-7). The critical physiologic anti-thromboticrole of protein S is revealed by the massive thrombotic complicationssuffered by infants homozygous for protein S deficiency (8,9). Inadults, mild heterozygous deficiencies in protein S are reported to beassociated with a risk for venous and arterial thrombosis (10-13),ischemic stroke (14,15), and cerebral thrombophlebitis (16,17).

In addition to its anticoagulant activity, protein S binds to vascularcells and is a potent mitogen (18-20). A structural homolog of proteinS, the growth arrest specific gene-6 (gas6), is a survival factor (21).Gas6 rescues cells from apoptosis induced by serum withdrawal (22-24).It has been suggested that both protein S and gas6 are ligands for theTyro3/Axl family of receptor tyrosine kinases (25). But the extent towhich protein S functions in vivo as a ligand for Tyro3/Axl receptorswas unclear (26) until the present results were obtained.

Ischemic strokes in humans are due to thrombotic or thromboembolicvascular occlusions (27) resulting in post-ischemic neurodegenerativedisorder. Protein S had significant anti-thrombotic activity in a rabbitmodel of peripheral arterial thrombosis (28), but its potential forstroke therapy has not been explored. In contrast to a fibrinolyticagent, e.g., tissue plasminogen activator (tPA) which may predispose toCNS bleeding (29) and is neurotoxic (30,31), elevated levels of bovineprotein S in rabbits did not cause bleeding (28). No neuroprotection wasobserved.

We determined whether protein S may control ischemic brain damage byprotecting cells of the brain (e.g., neurons, brain endothelial cells,vascular smooth muscle cells of brain vasculature, pericytes,astrocytes, microglia, oligodendrocytes, and stems cells includingneuronal or oligodendrocyte precursors) from ischemic/hypoxic injury;promoting anticoagulation; controlling cerebrovascular thrombosis; orcombinations thereof.

Hereditary protein S deficiency is an autosomal dominant disorder thatis associated with a risk of recurrent and inappropriate clot formation.Most likely consequences are venous thrombosis and pulmonary embolism,but protein S deficiency may also predispose patients to arterialthrombotic disease. Few homozygous or compound heterozygous subjectshave been reported. Such a genotype may be incompatible with survival toadulthood without treatment because of the development of severe purpurafulminans shortly after birth.

While protein S deficiency in the general population is relatively rare(up to a few percent), it is found in up to 10% of young patients withvenous thrombosis. Many other circumstances may lead to acquired proteinS deficiency such as oral anticoagulant therapy, oral contraception,liver disease, nephrotic syndrome, disseminated intravascularcoagulation, and pregnancy. Other factors that affect protein S activityare gender (women have a lower protein S level than men) and age (totalprotein S level increases with age in women due to their hormonalstatus). Total and free protein S levels are also positively correlatedwith triglyceride and cholesterol levels.

In neurodegenerative diseases, neuronal cells degenerate to bring aboutdeterioration of cognitive function. A variety of diseases andneurological deficiencies may bring about such degeneration includingAlzheimer's disease, Huntington disease or chorea, hypoxia or ischemiacaused by stroke, cell death caused by epilepsy, amyotrophic lateralsclerosis, mental retardation and the like, as well as neurodegenerativechanges resulting from aging.

The neuroprotective activity of protein S and its functional variantsmay also be obtained by increasing its biological activity (e.g., genetherapy, gene activation), decreasing the biological activity of aninhibitor (e.g., reducing C4b binding protein amount or activity), andother methods of altering protein S activity.

The present invention is useful for treating many clinical conditionsinvolving inflammatory processes. For example, inflammatory boweldisease including Crohn's disease and ulcerative colitis are spontaneouschronic inflammations of the gastrointestinal tract which involveactivation of inflammatory cells whose products cause tissue injury.Neutrophils, eosinophils, mast cells, lymphocytes, and macrophages maycontribute to the inflammatory response.

The present invention is also directed to treatment of systemic shockand many resultant clinical conditions associated therewith. Systemicshock often occurs as a complication of severe blood loss, severelocalized bacterial infection, or ischemia/reperfusion trauma and it isa major cause of death in intensive care units. Many cases of septicshock are induced by endotoxins (i.e., lipopolysaccharides or LPS) fromgram negative bacilli or toxins (i.e., toxic shock toxin 1) fromgram-positive cocci bacteria. The release of LPS in the bloodstreamcauses release of inflammatory mediators (e.g., cytokines, plateletactivating factor, complement, leukotrienes, oxygen metabolites, and thelike) which cause myocardial dysfunction, vasodilation, hypotension,endothelial injury, leukocyte adhesion and aggregation, disseminatedintravascular coagulation, adult respiratory distress syndrome (ARDS),or failure of liver, kidney, or central nervous system (CNS). Shock dueto blood loss also involves inflammatory mediator release. In each case,inflammatory responses are induced at the original site of trauma, andalso in the vasculature and remote vascularized sites.

Myocardial ischemia is associated with activation of the complementsystem which further promotes cardiac injury with the enhancement of aseries of inflammatory events. Life threatening local and remote tissuedamage occurs during surgery, trauma, and stroke when major vascularbeds are deprived for a time of oxygenation (ischemia) then restoredwith normal circulation (reperfusion). Reperfusion injury ischaracterized by vascular permeability leading to edema and infiltrationof inflammatory cells. Neutrophils contribute significantly toreperfusion damage by generating oxidants or releasing proteases thatdamage the microvasculature or adjacent tissue. Cell death and tissuedamage due to complement and inflammatory cell mechanisms lead to organfailure or decreased organ function. The activation of mediators by alocal injury can also cause a remote injury to highly vascularizedorgans. The compositions and methodologies of the present invention areuseful in the treatment of such injury.

Inflammatory response damage also occurs in glomerulonephritis as wellas tubule disease. Infiltration of inflammatory cells (especiallymacrophages) is linked to proteinuria accompanied histologically byhypercellularity and crescent formation in glomeruli. Over a longerterm, the infiltration of inflammatory cells is associated withaccumulation of extracellular matrix and sclerosis and chroniccompromise of renal function. The present invention is also efficaciousin treating glomerulonephritis and tubule disease.

There are many other disease and pathological conditions which benefitfrom the methodologies of the present invention such as for example,coronary arterial occlusion, cardiac arrhythmias, congestive heartfailure, cardiomyopathy, bronchitis, acute allergic reactions andhypersensitivity, neurotrauma, graft/transplant rejection, myocarditis,insulin dependent diabetes, and stroke. Stroke involves a very stronginflammatory response, that in part may be responsible for neuronaldamage directly by allowing leukocytes to enter the extravascularregions of the brain and destroy normal brain cells and neurons, andindirectly by obstructing microvessels and stopping blood flow due tothe procoagulant effects of inflammation. These intravascular andextravascular processes may require adhesion molecules and cytokinesthat are direct or indirect targets of cellular interactions which areindependent of anticoagulant effects.

In addition to treating patients suffering from the trauma resultingfrom heart attack, patients suffering from actual physical trauma couldbe treated in order to relieve the amount of inflammation and swellingwhich normally result after an area of the body is subjected to severetrauma. Also, patients suffering from hemorrhagic shock could be treatedto alleviate inflammation associated with restoring blood flow. Otherdisease states which might be treated using formulations of theinvention include various types of arthritis, various chronicinflammatory conditions of the skin, insulin-dependent diabetes, andadult respiratory distress syndrome. After reading the presentdisclosure, those skilled in the art will recognize other disease statesand/or symptoms which might be treated and/or mitigated by the presentinvention.

Some examples of arterial thrombosis where protein S alone or incombination with a thrombolytic agent, anticoagulant, anti-plateletagent, or anti-inflammatory agent is useful include the followingclinical settings: i) acute arterial thrombotic occlusion includingcoronary, cerebral, or peripheral arteries; ii) thrombotic occlusion orrestenosis after angioplasty; iii) reocclusion or restenosis afterthrombolytic therapy; and iv) venous thrombotic occlusion. Thrombolyticagents such as t-PA salvage ischerhic tissue when used within hours ofacute heart attack or stroke by re-establishing blood flow in theoccluded artery. Between one-fourth and one-third of patients who havesuccessful thrombolytic reperfusion of occluded coronary arteriessubsequently undergo reocclusion after discontinuing t-PA infusion. Thiscomplication occurs despite full-dose heparin therapy. The presentinvention may have greater efficacy than heparin in preventingreocclusion. Problems with thrombolytic therapy with t-PA includeneurotoxicity and killing of neurons. The addition of protein S mightreduce or prevent such unwanted consequences. v) Small and large calibervascular graft occlusion. Vascular grafts of small caliber, i.e., 3-/mmdiameter, have a high frequency of thrombotic occlusion. Protein S aloneor in combination with a thrombolytic agent is useful to preventocclusion. vi) Hemodialysis. The prosthetic surfaces and flow design ofall hemodialyzers are thrombogenic. Currently heparin is infused duringdialysis. However, heparin is only partially effective, thereby limitingthe reuse of dialyzers. Also, heparin has a number of troublesome sideeffects and complications. vii) Cardiopulmonary bypass surgery. Toprevent thrombus formation in the oxygenator and pump apparatus, heparinis currently used. However, it fails to inhibit platelet activation andthe resultant transient platelet dysfunction which predisposes tobleeding problems post-operatively. viii) Left ventricular cardiacassist device. This prosthetic pump is highly thrombogenic and resultsin life threatening thromboembolic events—complications that are onlypartially reduced by conventional anticoagulants (heparin or coumarindrugs). ix) Total artificial heart and left ventricular assist devices.x) Other arterial thrombosis. Protein S is useful for arterialthrombosis or thromboembolism where current therapeutic measures areeither contraindicated or not effective. For example, protein S isuseful for treating acute pre-or post-capillary occlusion, includingtransplantation, retinal thrombosis, or microthrombotic necrosis of anyorgan complicating infections, tumors, or coumarin treatment.

In another embodiment, the present invention provides methods forprotecting cells of the nervous system from cell death in a subjecthaving or at risk of disease or another pathological condition. Themethod includes administering an effective amount of protein S to thesubject to provide neuroprotection. Examples of such disorders include,but are not limited to, stroke, Alzheimer's disease, Huntington disease,ischemia, epilepsy, amyotrophic lateral sclerosis, mental retardationand aging. One “having or at risk of having” an inflammatory vasculardisease as described herein is a subject either exhibiting symptoms ofthe disease or diagnosed as being at risk for developing the disease.Such subjects include those having undergone or preparing for surgicalprocedures as described below.

In yet another embodiment, the invention provides methods for reducinginflammation in a subject having or at risk of having aneuropathological disorder. The method includes administering ananti-inflammatory effective amount of protein S to the subject, therebyreducing neurological inflammation in the subject. The methodologies ofthe present invention may also be efficacious in treating multiplesclerosis (MS) in addition to neuropathologies described above. MS isoften characterized by the penetration of the blood-brain barrier bycirculating leukocytes, leading to demyelination in various parts of thebrain, impaired nerve conduction and, ultimately, paralysis.

The term “neuroprotective” is used to denote protection of any type ofcell of the nervous system including neurons, brain endothelial cells,brain vascular smooth muscle cells, pericytes, astrocytes,oligodendrocytes, stem cells including neuronal and oligodendrocyteprecursors, and microglia from cellular stress, injury, and/or celldeath, including ischemia and hypoxia.

The term “neurodegenerative disease” is used to denote conditions whichresult from loss of neurons, neuronal cell injury or loss, and/or injuryof other types of brain cells such as oligodendrocytes or brainendothelial cells and/or other vascular cells, but not limited to anycell type in the nervous system which may bring about deterioration ofmotor or sensory functions of the nervous system, cognitive function,higher integrative intellectual functions, memory, vision, hearing etc.Such degeneration of neural cells may be caused by Alzheimer's diseasecharacterized by synaptic loss and loss of neurons; Huntington diseaseor chorea; by pathological conditions caused by temporary lack of bloodor oxygen supply to the brain, e.g., brought about by stroke; byepileptic seizures; due to chronic conditions such as amyotrophiclateral sclerosis, mental retardation; as well as due to normaldegeneration due to aging. It should be noted that diseases such asstroke and Alzheimer's have both a neurodegenerative and an inflammatoryvascular component and thus are treated by the methods of the invention.

One aspect of the invention includes the neuroprotective activity ofprotein S. The term “neuron” includes hundreds of different types ofneurons, each with distinct properties. Each type of neuron produces andresponds to different combinations of neurotransmitters and neurotrophicfactors. Neurons are thought not to divide in the adult brain, nor dothey generally survive long in vitro. The method of the inventionprovides for the protection from cell death or injury of neurons fromvirtually any region of the brain and spinal cord. Neurons include thosein embryonic, fetal or adult neural tissue, including tissue from thehippocampus, cerebellum, spinal cord, cortex (e.g., motor orsomatosensory cortex), striatum, basal forebrain (e.g., cholenergicneurons), ventral mesence-phalon (e.g., cells of the substantia nigra),and the locus ceruleus (e.g., neuro-adrenaline cells of the centralnervous system).

Genetics and Structure of Protein S

The human genome contains two genes: PROS1 is functional and encodesprotein S and PROS2 is a pseudogene. Both genes are located onchromosome 3 at 3p11.1-3q11.2 and are linked within 4 cM. The PROS1 geneoccupies about 80 kb of DNA with 15 exons. PROS1 and PROS2 are 97% and95% identical between corresponding exons and introns, respectively.PROS2 lacks exon 1 and contains multiple base changes in the codingportions with termination codons at amino acid residues 61, 299, 410 and522 a frameshift mutation in exon 10. Three mRNA species are transcribedfrom PROS1 and then translated into human protein S. The major mRNAspecies is about 4 kb. Three frequent polymorphisms have been describedin the protein S gene: the first is located in the coding region (Pro626 encoded either by CCA or by CCG) and the other two are located innoncoding regions (a C to T transition in intron 5 which is four basesdownstream of exon 11, and a C to A transversion which is 520 basesdownstream of the Stop codon).

The intron-exon organization of the gene for protein S reflects itsmodular structure. The first eight exons encode structural/functionaldomains also found in other vitamin K-dependent coagulation proteins(except for exon IV, coding for the thrombin-sensitive loop) and havebeen placed upstream of the ancestral gene of a steroid hormone bindingprotein. The 3′ part of exon 1 codes for the signal peptide, exon 2 forthe propeptide and the GLA-domain, exon 3 for the helical stack domain,exon 4 for the thrombin-sensitive loop, exons 5 to 8 for four epi-dermalgrowth factor-like domains, and exons 9 to 14 and the first 161 bp ofexon 15 for the sex hormone-binding globulin-homologous domain. At leastpartial sequences for protein S from human, monkey, mouse, rat, rabbitand cow are known. After alignment, they are about 59% identical at theamino acid level.

The plasma concentration of protein S is about 25 mg/L (about 0.33 μM).The protein functions as a non-enzymatic cofactor to activated protein C(APC) in the proteolytic degradation of factors Va and VIIIa. Protein Sincreases 10-fold the affinity of APC for negatively chargedphospholipids. The two proteins form a putative 1:1 complex on lipidsurfaces such as platelets. Protein S has a direct APC-independentanticoagulant activity by inhibition of prothrombinase activity and offactor X activating complex by binding to factor VIII. The importance ofthese properties in physiological anticoagulant mechanisms remains to bedemonstrated. Protein S circulates in human plasma in two forms: about40% free and about 60% bound to a regulator of the classical complementpathway, C4b-BP. Only the free protein S (about 120 nM) has cofactoractivity for APC. The plasma concentration of C4b-BP is about 150 mg/L(about 0.26 μM). Interaction between protein S and C4b-BP isnon-covalent and reversible. Protein S interacts with the β-chain ofC4b-BP while the α-chains of C4b-BP are devoted to binding thecomplement protein C4b. Thus, only C4b-BP isoforms containing a β-chain(representing about 80% of circulating C4b-BP) are able to bind proteinS. In the presence of calcium ions, the dissociation constant isapproximately 5×10⁻¹⁰ M and all β-chain containing C4b-BP is linked toprotein S. In healthy individuals, the concentration of free protein Sis largely determined by the concentration of C4b-BPβ+ and correspondsto the molar excess of protein S over C4b-BPβ+.

Protein S in its mature form is a single-chain glycoprotein of 635 aminoacids resulting from post-translational modification of a 676 amino acidprecursor. It has three glycosylation sites (Asn 458, 468 and 489) andseven domains with different functional or structural roles. The signalpeptide (residues −41 to −18) inserts into the rough endoplasmicreticulum and drives membrane translocation; the propeptide (residues−17 to −1) is necessary for carboxylase recognition and y-carboxylation.These two domains are released by a cleavage reaction before secretion.The mature N-terminal part of the protein is composed of a GLA-domain(residues 1 to 37) containing 11 γ-carboxyglutamic acids which bindmultiple calcium ions. The resulting stabilized structure has a highaffinity for negatively-charged phospholipid membranes. The GLA-domainis followed by a short helical stack (residues 38 to 45) with arelatively high content of aromatic residues.

Whereas the above domains are present in all vitamin K-dependentproteins, the thrombin-sensitive region (residues 46 to 72) is foundonly in protein S. This domain contains two Cys residues (47 and 72)linked by a disulfide loop in which three peptide bonds are sensitive tothrombin proteolysis and it has recently been shown that circulatingcleaved protein S is cleaved after Arg60, a site already described assensitive to factor Xa cleavage. Whatever enzyme is responsible for thein vivo cleavage of protein S, the GLA-domain remains linked to the restof the molecule by the disulfide bond. Since the GLA domain can nolonger adopt the calcium-dependent conformation required for biologicalactivity, protein S cannot bind to phospholipids at physiologicalcalcium ion concentration and APC cofactor activity is lost. Therefore,this mutation may be used to separate APC cofactor activity from otheractivities of protein S (e.g., neuroprotective activity). These findingsalso suggest that the thrombin-sensitive loop interacts with APC and isinvolved in GLA-domain folding. Four epidermal growth factor-likedomains (residues 76 to 242) are adjacent to the thrombin-sensitiveregion. EGF1 contains a β-hydroxy-lated Asp residue while the otherthree contain β-hydroxylated Asn. The EGF domains contain high-affinitycalcium ion binding sites. The carboxy-terminal half of protein S is alarge module homologous to sex hormone binding globulin. It contains twosmall disulfide loops formed by internal disulfide bonds. This moduledoes not bind steroids, but contains at least two potential interactionsites with C4b-BP: residues 420 to 433 and residues 583 to 635.

Mutations in the protein S gene may be classified as qualitative orquantitative. A qualitative deficiency (type II) results in decreaseprotein S activity associated with normal levels of total or freeprotein S. Quantitative deficiencies have reductions in both total andfree protein S (type I) or only free protein S (type III).

Frameshift mutations included 27 different insertions or deletionssmaller than seven bases. Assay of protein S in plasma showed type Ideficiency. Other quantitative defects resulted from insertions,deletions, splice site mutations, frameshift mutations, and missensemutations. Only seven different nucleotide substitutions are known to beresponsible for type II deficiency. Five are missense mutations(Arg-2Leu, Arg-1His, Lys9Glu, Thr103Asn, Lys155Glu) with three beinglocated in the pro-peptide or the GLA domain. The plasma phenotype ofthe patient with the Thr103Asn mutation supports the role of this aminoacid in the interaction with APC. Two splice site mutations were alsoassociated with type II deficiency. One activated a cryptic splice site(intron g, AT, exon 8-2) and results in a deletion of two amino acidsfrom the protein (IIe-Asp 203-204). The other resulted in twoalternative splice transcripts, lacking either exon 5 or both exons 5and 6. An EGF1-lacking protein S species, corresponding to the exon5-lacking transcript, was detected in the patient's plasma.

Three frequent polymorphisms and 18 rare polymorphisms do not appear tohave an effect on protein S activity. They include missense changes(Pro35Leu, Arg192Lys, Thr477Met); silent changes which did not changethe encoded amino acid (Leu-30Leu; Pro35Pro; IIe30311e, Gly418Gly); andthose that do not cose-gregate with the protein S deficiency (5′UT TCexon 1-286; intron a, AG, exon 1 +7; intron a, del ATT, exon 2 −7;intron b, GA, exon 2 +5; intron g, GA, exon 8 −20, Arg49His, Thr57Ser,Met344Val, Ile518Met). Variation at one of the three glycosylation sitesN-X-SIT (Ser460Pro) results in loss of the sugar modification, but nofunctional consequence of this change has been unequivocallydemonstrated.

An electronic database is available of more than 100 different mutationsand polymorphisms of human protein S (see Gandrille et al. Thromb.Haemost. 77:1201-1214, 1997). It is preferred that the protein S orfunctional variant thereof be derived from the same species as theorganism being treated.

“Protein S” refers to native genes and proteins belonging to this familyas well as variants thereof (e.g., mutations and polymorphisms found innature or artificially designed). The chemical structure of the genesand proteins may be a polymer of natural or non-natural nucleotidesconnected by natural or non-natural covalent linkages (i.e.,polynucleotide) or a polymer of natural or non-natural amino acidsconnected by natural or non-natural covalent linkages (i.e.,poly-peptide). See Tables 1-4 of WIPO Standard ST.25 (1998) for anonlimiting list of natural and non-natural nucleotides and amino acids.See Tables 1-4 of WIPO Standard ST.25 (1998) for a nonlimiting list ofnatural and non-natural nucleotides and amino acids. Protein S genes andproteins may be recognized as belonging to this family by comparison tothe human homologs PROS1 and PROS2, use of nucleic acid binding (e.g.,stringent hybridization conditions of 400 mM NaCl, 40 mM PIPES pH 6.4, 1mM EDTA, at 50° C. or 70° C. for an oligonucleotide; 500 mM NaHPO₄ pH7.2, 7% SDS, 1% BSA, 1 mM EDTA, at 45° C. or 65° C. for a polynucleotideof 50 bases or longer; and appropriate washing) or protein binding(e.g., specific immunoassay under stringent binding conditions of 50 mMTris-HCl pH 7.4, 500 mM NaCl, 0.05% TWEEN 20 surfactant, 1% BSA, at roomtemperature and appropriate washing); or computer algorithms (Doolittle,Of URFS and ORFS, 1986; Gribskov & Devereux, Sequence Analysis Primer,1991; and references cited therein). For example, washing may beinitiated at an ionic strength, pH, and temperature equivalent to thehybridization/binding conditions (with or without blocking agents and/orsurfactants), then decreasing the salt concentration or increasing thetemperature with one or more changes of washing solution until thedesired degree of specificity is achieved.

A “mutation” refers to one or more changes in the sequence ofpoly-nucleotides and polypeptides as compared to native protein S, andhas at least one function that is more active or less active, anexisting function that is changed or absent, a novel function that isnot naturally present, or combinations thereof. In contrast, a“polymorphism” also refers to a difference in its sequence as comparedto native protein S, but the changes do not necessarily have functionalconsequences. Mutations and polymorphisms can be made by geneticengineering or chemical synthesis, but the latter is preferred fornon-natural nucleotides, amino acids, or linkages. Fusions of domainslinked in their reading frames are another way of generating diversityin sequence or mixing-and-matching functional domains. For example,homologous protein C and protein S work best together and this indicatesthat their sequences may have coevolved to optimize interactions betweenthe enzyme and its cofactor. Exon shuffling or gene shuffling techniquesmay be used to select desirable phenotypes in a chosen background (e.g.,combining sequence changes that confer loss of glycosylation at Asn458and APC cofactor activity, hybrid human/mouse sequences which locate thespecies determinants).

Percentage identity between a pair of sequences may be calculated by thealgorithm implemented in the BESTFIT computer program (Smith & Waterman.J. Mol. Biol. 147:195-197,1981; Pearson, Genomics 11:635-650,1991).Another algorithm that calculates sequence divergence has been adaptedfor rapid database searching and implemented in the BLAST computerprogram (Altschul et al., Nucl. Acids Res. 25:3389-3402, 1997). Incomparison to the human sequence, the protein S polynucleotide orpolypeptide may be only about 60% identical at the amino acid level(protein S from different mammals are only about 59% identical), 70% ormore identical, 80% or more identical, 90% or more identical, or greaterthan 95% identical.

Conservative amino acid substitutions (e.g., Glu/Asp, Val/lle, Ser/Thr,Arg/Lys, Gln/Asn) may also be considered when making comparisons becausethe chemical similarity of these pairs of amino acid residues areexpected to result in functional equivalency in many cases. Amino acidsubstitutions that are expected to conserve the biological function ofthe polypeptide would conserve chemical attributes of the substitutedamino acid residues such as hydrophobicity, hydrophilicity, sidechaincharge, or size. Functional equivalency or conservation of biologicalfunction may be evaluated by methods for structural determination andbioassay.

The codons used may also be adapted for translation in a heterologoushost by adopting the codon preferences of the host. This wouldaccommodate the translational machinery of the heterologous host withouta substantial change in chemical structure of the polypeptide. Forexample, a mammalian protein S or variant thereof may have its codonsaltered for translation in a bacterial or fungal host.

Protein S and variants thereof (i.e., deletion, domain shuffling orduplication, insertion, substitution, or combinations thereof) may beused to determine structure-function relationships (e.g., alaninescanning, conservative or nonconservative amino acid substitution). Forexample, protein S folding and processing, protein S secretion, proteinS binding to phospholipids and other proteins, any of the biologicalactivities described herein, or combinations thereof may be related tochanges in the amino acid sequence. See Wells (Bio/Technology 13:647651,1995) and U.S. Pat. No. 5,534,617. Directed evolution by directed orrandom mutagenesis or gene shuffling using protein S may be used toacquire new and improved functions in accordance with selectioncriteria. Mutant and polymorphic variant polypeptides are encoded bysuitable mutant and polymorphic variant polynucleotides.Structure-activity relationships of protein S may be studied (i.e., SARstudies) using variant polypeptides produced with an expressionconstruct transfected in a host cell with or without expressingendogenous protein S. Thus, mutations in discrete domains of protein Smay be associated with decreasing or even increasing activity in theprotein's function.

Formulations and Their Administration

Protein S or variants thereof may be used to formulate pharmaceuticalcompositions with one or more of the utilities disclosed herein. Theymay be administered in vitro to cells in culture, in vivo to cells inthe body, or ex vivo to cells outside of a subject which may then bereturned to the body of the same subject or another. The cells may beremoved from, transplanted into, or be present in the subject.

Use of compositions which further comprise a pharmaceutically acceptablecarrier and compositions which further comprise components useful fordelivering the composition to a subject's brain are known in the art.Addition of such carriers and other components to the composition of theinvention is well within the level of skill in this art.

A pharmaceutical composition may be administered as a formulation whichis adapted for direct application to the central nervous system, orsuitable for passage through the gut or blood circulation.Alternatively, pharmaceutical compositions may be added to the culturemedium. In addition to active compound, such compositions may containpharmaceutically-acceptable carriers and other ingredients known tofacilitate administration and/or enhance uptake. It may be administeredin a single dose or in multiple doses which are administered atdifferent times.

Pharmaceutical compositions may be administered by any known route. Byway of example, the composition may be administered by a mucosal,pulmonary, topical, or other localized or systemic route (e.g.,parenteral). “Parenteral” includes subcutaneous, intradermal,intramuscular, intravenous, intra-arterial, intrathecal, and otherinjection or infusion techniques, without limitation. In particular,achieving an effective amount of protein S in the central or peripheralnervous system may be desired. This may involve a depot injection intoor surgical implant within the brain. Intravenous administration may beused for stroke and intra-arterial administration may be used duringneurosurgery.

Suitable choices in amounts and timing of doses, formulation, and routesof administration can be made with the goals of achieving a favorableresponse in the subject (i.e., efficacy), and avoiding undue toxicity orother harm thereto (i.e., safety). Therefore, “effective” refers to suchchoices that involve routine manipulation of conditions to achieve adesired effect (e.g., neuroprotection; anti-thrombotic activity;anti-inflammatory activity; inhibition of apoptosis; or preventing theinjury caused by ischemia, hypoxia, re-oxygentation, or the like).

A bolus of the formulation administered only once to a subject is aconvenient dosing schedule although achieving an effective concentrationof protein S in the brain may require more frequent administration.Alternatively, an effective dose may be administered every other day,once a week, or once a month. Dosage levels of active ingredients in apharmaceutical composition can also be varied so as to achieve atransient or sustained concentration of the compound or derivativethereof in a subject and to result in the desired therapeutic response.But it is also within the skill of the art to start doses at levelslower than required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.

The amount of compound administered is dependent upon factors such as,for example, bioactivity and bioavailability of the compound (e.g.,half-life in the body, stability, and metabolism); chemical propertiesof the compound (e.g., molecular weight, hydrophobicity, andsolubility); route and scheduling of administration; and the like. Itwill also be understood that the specific dose level to be achieved forany particular subject may depend on a variety of factors, includingage, health, medical history, weight, combination with one or more otherdrugs, and severity of disease.

The term “treatment” refers to, inter alia, reducing or alleviating oneor more symptoms of disease or another pathological condition in asubject. This includes therapy of an affected subject or prophylaxis ofa subject at risk. For a given subject, improvement in a symptom, itsworsening, regression, or progression may be determined by an objectiveor subjective measure. Treatment may also involve combination with otherexisting modes of treatment and agents (e.g., protein C, activatedprotein C, other anti-thrombotic agents, steroidal or nonsteroidalanti-inflammatory agents). Thus, combination treatment may be practiced.

EXAMPLES

The effects of purified human plasma-derived or recombinant protein Swas examined in a murine in vivo model of focal ischemic stroke and anin vitro neuronal hypoxic/re-oxygenation injury. Protein S significantlyimproved motor neurological function after stroke and reduced braininfarction and edema in a dose-dependent manner. At higherconcentrations protein S enhanced post-ischemic reperfusion and reducedbrain fibrin and neutrophils deposition. In vitro protein S protectedcultured cortical neurons from hypoxia/re-oxygenation-induced apoptosis.Protein S may be a prototype of a new class of neuroprotective agentswith combined anti-thrombotic, anti-inflammatory and direct cellularneuroprotective effects to treat disease associated with ischemia,hypoxia, and other re-oxygenation injury (e.g., stroke) as well assimilar diseases and other pathological conditions.

Animals. Procedures were approved by the University of Rochester'sInstitutional Animal Care and Use Committee. Male C57BL/6 mice (23-26gm) were anesthetized with an intraperitoneal injection of ketamine (100mg/kg) and xylazine (10 mg/kg). Animals were allowed to breathspontaneously. Rectal temperature was maintained at 37° C.±1° C. Theirright femoral arteries were cannulated for monitoring of blood pressureand blood analysis.Stroke model. A modification of the intravascular, middle cerebralartery (MCA) occlusion technique (33,34) was used to induce stroke. Anon-siliconized non-coated 6-10 mm±1 mm long prolene suture with arounded tip (diameter 0.20 mm) was advanced into the internal carotidartery to occlude the MCA for 1 hr followed by 23 hr of reperfusion.

Protein S, human plasma-derived (0.2, 0.5 or 2 mg/kg), human recombinantprotein S (2 mg/kg) or vehicle were administered intravenously 10 minafter the MCA occlusion (n=6 per group). Protein S was purified aspreviously described (4).

Protein S was given at 10 min after the induction of stroke when bloodflow was at a minimum to give a reasonable test of the bioactivity ofprotein S during ischemia in the murine model (33). In this regard, wenote that the time course of pathophysiological changes in the presentmodel is different from human strokes and the occlusion in this model isremoved after one hour. Moreover, in the clinical situation in humans,spontaneous reopening of major occluded blood vessels in patients withischemic stroke does not typically happen within one hour after theinsult (27).

Cerebral blood flow (CBF) was monitored by Laser Doppler Flowmetry (LDF,Transonic Systems) (33,34). LDF probes (0.8 mm diameter) were positionedon the cortical surface 2 mm posterior to the bregma, both 3 mm and 6 mmto each side of midline. The procedure was considered successful if ≧80%drop in CBF was observed immediately after placement of the suture. Headtemperature was monitored with a 36-gauge thermocouple probe in thetemporalis muscle (Model 9000, Omega, Conn.).

Neurologic examinations were performed at 24 hr and scored (33): noneurologic deficit 0, failure to fully extend left forepaw 1, turning toleft 2, circling to left 3, unable to walk spontaneously 4, andstroke-related death 5.

Arterial blood gases (pH, PaO₂, PaCO₂) were measured before and duringMCA occlusion using ABL 30 Acid-Base Analyzer (Radiometer).

Unfixed 1 mm coronal brain slices were incubated in 2% TTC in phosphatebuffer (pH 7.4). Serial coronal sections were displayed on a digitizingvideo screen (Jandel Scientific). Brain infarction and edema volume werecalculated using Swanson correction (33,34).

Histopathology and fibrin detection. Leukocytes were stained usinganti-CD11b antibody (DAKO) (1:250 dilution) directed against leukocyteMac-1 (33). The number of CD11b positive cells in tissue was given permm². Previous study has demonstrated that the number of CD11b positivecells and dichloracetate esterase (a specific marker for neutrophils)positive cells in the murine model of stroke is identical (33). Countingwas performed in ten random fields in the ischemic hemisphere by twoindependent observers blinded to the specimen source or timing. Routinecontrols included deletion of primary antibody, deletion of secondaryantibody and/or the use of an irrelevant primary antibody. The amount offibrin was quantified in 1 mm thick brain hemisections by Westernblotting using anti-fibrin II antibody (NYB-T2G1, Accurate Chemical)(1:500 dilution) as described (33,34).Protein S ELISA Assay. The amount of human protein S in plasma of miceat 1 hr after they received protein S injections (0.2, 0.5, 2.0 or 6.0mg/kg) was quantitated by ELISA as follows. Nunc Maxisorp microplateswere coated with 20 μg/ml of polyclonal rabbit purified IgG anti-proteinS (DAKO Corp.) in 0.1 M Na carbonate, pH 9.0 (150 μl/well) overnight at10° C. and then blocked with 200 μl buffer/well containing 50 mM Tris100 mM NaCl, pH 7.4, 2% BSA for 2 hr. Aliquots (150 μl) of plasmadiluted 1/400 and 1/1600 in 50 mM Tris, 100 mM NaCl, 0.02% Tween-20,0.5% BSA were added to wells and incubated for 2 hr. Following washingwith TBS, 0.02% Tween-20, polyclonal HRP-labeled rabbit antibody (5μg/ml, DAKO) was used with OPD substrate (Sigma Chemical) to detectbound protein S. Standard curves, valid for 5 to 125 ng/ml protein S,were made with dilutions ( 1/200 to 1/6400) of pooled normal humanplasma (assumed to contain 25 μg/ml protein S, George King Inc.). Aplasma pool from 10 normal male mice gave no signal in this assaywhereas the same plasma containing purified human protein S (finalconcentration of 25 μg/ml) gave a standard curve indistinguishable frompooled human plasma.Cell Culture. Primary neuronal cortical cultures were established asdescribed (35). In brief, cerebral cortex was dissected from fetalC56BL/6 mice at 16 days of gestation, treated with trypsin for 10 min at37° C. and dissociated by trituration. Dissociated cell suspensions wereplated at 5×10⁵ cells per well on 12-well corning tissue culture dishescoated with poly-D-lysine, in serum-free Neurobasal medium plus B27supplement (GIBCO BRL). The absence of astrocytes was confirmed bynegative staining for the glial fibrillary acidic protein. Cultures weremaintained in a humidified 5% CO₂ incubator at 37° C. for 5 days beforetreatment. To induce hypoxic re-oxygenation injury, five-day-oldcultures were treated first for 12 hr with 95% N₂/5% CO₂ in DMEMserum-free medium without glucose, and next for 12 hr exposed tonormoxic conditions and medium containing 5 mM glucose (36). Protein S,human plasma-derived (1 nM to 1,000 nM) protein S, or recombinantprotein S was added to the medium throughout the entire 24 hr of thestudy. Cultures were next fixed for 10 min with 4% formaldehyde in PBSat 4° C. and double stained with Hoechst 33258 (1 μg/ml) and TUNEL(terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labeling)to determine nuclear morphological changes and the number of apoptoticcells.Oxidative Stress Model. Human microvascular brain endothelial cells(MBEC) were isolated by biopsy and cultured using methods similar tothose previously reported by Mackic et al. (J. Clin. Invest.102:734-743, 1998). Briefly, brain tissue was cut into small pieces, andthen mechanically dissociated using a loose-fitting cell homogenizer inRPMI 1640 with 2% fetal calf serum (FCS) and penicillin/streptomycin.The homogenate was then fractionated over 15% dextran by centrifugationat 10,000 g for 10 min to obtain a brain microvessel pellet.Microvessels were further digested with 1 mg/ml of collagenase/dispaseand 5 μl/ml of DNase in FCS-enriched medium for 1 hr at 37° C. This cellsuspension was centrifuged at 1000 g for 5 min, and the cell pellet wasplated on fibronectin-coated flasks in RPMI 1640 with 10% FCS, 10%NuSerum, endothelial cell growth factors, nonessential amino acids,vitamins, and penicillin/streptomycin as a primary culture.

The P0 primary cultures were grown to confluence, and sorted based onLDL binding using the Dil-Ac-LDL method following the manufacturer'sinstructions (Biomedical Technology). Briefly, cells were incubated withDil-Ac-LDL ligand for 4 hr at 37° C., trypsinized, and then separated byfluorescence activated cell sorting (FACS). Labeled and unlabeled humanumbilical vein endothelial cells (HUVEC) were used to set gating limitsas positive and negative controls, respectively. Unlabeled MBEC wereused to control for possible background staining or differences based oncell size. Positively sorted cells were plated on fibronectin- orcollagen-coated flasks in the medium described above. Cultures weregrown in 5% CO₂ and split 1:3 at confluency with collagenase/dispase.

Subconfluent brain endothelial cell cultures (3-4 days after subculture)were treated with H₂O₂ by adding it to the culture medium for 2 hr. Toinduce senescence sublethal doses of H₂O₂ were determined and selected.After treatment the cells were washed with PBS (37° C.) beforeharvesting, subculturing, or incubating with a fresh medium or 3-Dcollagen gels.

Statistics. Physiological variables, injury, infarction and edemavolumes were compared using ANOVA followed by Dunnett's multiplecomparisons test with the control group or Student's t-test when twogroups were compared. Non-parametric data (neurologic outcome scores)was subjected to the Chi-squared test with Fisher's transformation.

Animals treated with protein S had no significant differences in meanarterial blood pressure, PaO₂, PaCO₂, pH, hematocrit, head temperature,and blood glucose when compared with control animals. Protein Sadministration did not influence CBF under basal conditions. During MCAocclusion, the CBF in the control group dropped to 17-18% of baseline(p<0.001); treatment with protein S (0.2 mg/kg to 2 mg/kg) did notimprove the CBF during the occlusion phase (FIG. 1, Table 1). Duringpost-ischemic reperfusion, the CBF returned to 58-52% of baseline in thecontrol group (FIG. 1A, Table 1). Protein S at 2 mg/kg significantlyimproved the CBF during post-ischemic reperfusion by 21% to 26% (p<0.05;Table 1), but the effects of the lower doses of protein S were eithermarginal or lacking (see Table 1).

TABLE 1 CBF after MCA occlusion (60 min) followed by reperfusion.Occlusion Reperfusion Treatment 0-30 min 30-60 min 0-30 min 30-60 minVehicle 17.5 + 2.5 17.7 + 1.8 58.2 + 3.8 52.1 + 2.9 Protein S 2.0 mg/kg17.9 + 1.5 16.8 + 1.3 70.6 + 2.5* 65.8 + 3.4* 0.5 mg/kg 16.0 + 2.116.9 + 3.2 65.7 + 1.6* 58.5 + 3.3 0.2 mg/kg 15.3 + 3.5 16.0 + 2.3 60.1 +4.7 59.7 + 4.9CBF during MCA occlusion/reperfusion in control and protein S-treatedmice. Vehicle or protein S were given 10 min after initiation of MCAocclusion. CBF values (mean+SD) were averaged over studied period attime of occlusion or reperfusion and expressed as a percentage ofbaseline. *p<0.05

Control mice developed significant motor neurological deficit with ascore close to 4 (Table 2). At the lowest dose (0.2 mg/kg), protein Sreduced motor deficit and improved the average score by 1.4-fold, whileat higher doses at 0.5 mg/kg and 2 mg/kg protein S improvedsignificantly the motor score by 3.2-fold (Table 1).

TABLE 2 Motor neurological scores at 24 hr after MCAocclusion/reperfusion. No. of mice With score of Score Treatment 0 1 2 34 5 (mean ± SE) Vehicle 0 0 0 1 5 0 3.83 ± 0.17 Plasma-derived Protein S2.0 mg/kg 3 1 1 0 1 0 1.17 ± 0.65* 0.5 mg/kg 3 1 1 0 1 0 1.17 ± 0.65*0.2 mg/kg 3 1 0 0 1 0 1.00 ± 0.77*Protein S or vehicle were administered 10 min after stroke induction.*p<0.05 by Kruskal-Wallis test.

Protein S-treated animals were sacrificed at 24 hr to determine thevolume of brain injury. Protein S significantly reduced brain injuryvolume and edema volume in a dose-dependent manner by 35% and 43% at 0.2mg/kg (p<0.05), respectively, and by 59% and 62% at 2 mg/kg (p<0.01),respectively (FIGS. 2A and 2B). As shown for example in FIG. 2C, theinfarction area was significantly reduced in four out of the sevencoronal sections with 0.5 mg/kg of protein S. All control mice hadsignificant injury in the cortex and striatum on the side of theocclusion (FIG. 3); ≧50% of mice exhibited changes in the medialstriatum while <50% had changes in the dorsomedial and ventromedialcortex (FIG. 3A). Protein S (2 mg/kg) limited brain injury to a smallwell-localized area in the striatum and spared most of the brain (FIG.3B). Similar effects were obtained with recombinant protein S.

At 2 mg/kg, protein S reduced the amount of deposited fibrin in theischemic hemisphere by 40% (p<0.05; FIGS. 4A and 4B) and the number ofCD11 b-positive leukocytes by 53% (p<0.01; FIG. 4C).

To quantitate the circulating protein S levels in these studies, fourmice were each injected with each dose of protein S. Blood samples wereobtained an hour later and levels of human protein S in mouse plasmawere determined using an ELISA. The mean levels of circulating humanprotein S were 4.9, 11.0, 51.8 or 155 μg/ml for injections of 0.2, 0.5,2.0 or 6.0 mg/kg, respectively.

The direct effects of protein S in vitro on cultured mouse corticalneurons were studied. Neurons cultured under normoxic conditionsexhibited occasionally TUNEL-positive staining and chromatincondensation (FIG. 5A, left panels). In contrast, during ischemichypoxia/re-oxygenation injury, most of the cultured neurons wereTUNEL-positive and exhibited nuclear condensation and/or fragmentation(FIG. 5B, middle panels). In the presence of protein S there wasapproximately 70% reduction (p<0.05) in the number of apoptotic cells(FIG. 5A, right panels and FIG. 5B). Under the present experimentalconditions, protection of neurons from apoptotic death by protein S wastime-dependent and dose-dependent with the half-maximal effect EC₅₀ at75 nM (FIGS. 5C-5D).

Neuroprotective effects of plasma-derived human protein S on primaryhuman microvascular brain endothelial cells (MBEC) obtained frombiopsies and exposed to 500 μM H₂O₂ for 1.5 hr. In this oxidative stressmodel, about 50-70% of cells became apoptotic as shown by TUNEL andHoechst staining. Protein S administered two hours prior to oxidativestress reduced the number of TUNEL-positive cells in a dose-dependentmanner with an EC₅₀ of 200 nM. Next, it was demonstrated that IgGanti-annexin II (2.5 μg/ml) and IgG anti-Tyro3 (2.0 μg/ml) inhibitby >95% the anti-apoptotic effects of protein S. These findings indicatethat protein S acts as a cell survival factor in brain endotheliumexposed to oxidative damage, and that annexin II and Tyro3 are requiredfor its anti-apoptotic effects.

Data presented have demonstrated neuroprotective, anti-thrombotic, andanti-inflammatory effects of protein S in a murine in vivo model offocal ischemic stroke with reperfusion and direct neuronal protectiveeffects of protein S in an in vitro model of ischemia using murinecultured cortical neurons challenged by hypoxialaglycemia followed byre-oxygenation. In the stroke model, protein S reduced the motorneurological deficit, the infarction volume and the edema volume in adose-dependent manner. The effect of protein S on post-ischemic CBFduring reperfusion was significant with 2 mg/kg, but marginal with anintermediate dose (0.5 mg/kg) and/or absent with a low 0.2 mg/kg dose.It is noteworthy that the same low dose of protein S (0.2 mg/kg)significantly reduced the injury and edema volumes by 35% and 43%,respectively, in spite of the lack of an observable effect on CBF.

We studied protein S in a murine in vivo model of stroke and an in vitromodel of neuronal hypoxic/re-oxygenation injury. Animals receivedpurified human plasma-derived protein S or vehicle intravenously 10 minafter initiation of middle cerebral artery occlusion followed byreperfusion. Protein S at 0.2 to 2 mg/kg significantly improved themotor neurological deficit by 1.4- to 3.2-fold and reduced infarctionvolume by 35 to 59% and brain edema by 45 to 62% in a dose-dependentmanner. Protein S at 2 mg/kg improved the post-ischemic cerebral bloodflow by 21% to 26% and reduced brain fibrin deposition and infiltrationwith neutrophils by 40% and 53%, respectively. Intracerebral bleedingwas not observed with protein S. Protein S protected cultured neuronsfrom hypoxia/re-oxygenation-induced apoptosis in a dose-dependentmanner. Recombinant human protein S exerted similar protective effectsfrom hypoxia-induced damage as the plasma-derived protein S both in vivoand in vitro.

Significant obstructions in CBF in focal stroke might result frommicrovascular occlusions due to fibrin deposition, vascular accumulationof neutrophils and brain swelling (33,34). Previous studies reportedsignificant anticoagulant activity of protein S in vitro and in vivo(2-7,28). The present study confirmed reduced fibrin deposition andreduced infiltration of brain tissue with leukocytes in the presence ofprotein S. Protein S alleviated ischemic cerebral coagulopathy andreduced ischemic microvascular obstructions with blood cells, therebylimiting the development of brain thrombosis and contributing to therestoration of post-ischemic brain perfusion. However, thecerebroprotective effects were also observed with the lower doses ofprotein S, which apparently did not affect and/or improve significantlythe post-ischemic CBF. Therefore, in addition to anti-thromboticmechanisms of protein S during brain ischemia, we also considered otherpossible mechanisms including direct neuroprotective cellular effects.

Remarkably, our studies also demonstrated that protein S directlyprotects ischemic cultured neurons exposed to hypoxic/re-oxygenationinjury in vitro. In the presence of protein S the number of culturedneurons that were TUNEL-positive and exhibiting nuclear shrinkage,chromatin condensation and nuclear fragmentation were significantlyreduced in a dose-dependent manner. Under these conditions, protein Swas able to spare about 70% of neurons with an EC₅₀ of 75 nM. Cellbinding and mitogenic effects of protein S have been demonstrated invascular smooth muscle cells (18-20), while the anti-apoptotic effectsof gas6, a structural homolog of protein S, are well established(22-24). The molecular nature of a protein S receptor as a transmembranetyrosine kinase receptor has been debated (25,26). Direct anti-apoptoticactivity of protein S has not been previously described. Some studieshave suggested that both gas6 and protein S are ligands for theTyro3/Axl family of receptor tyrosine kinases (25). However, the role ofTyro3/Axl receptors has not been confirmed by others (20,26) and thenature of the receptor for protein S on neuronal cells was unknown. Ithas been also suggested that mitogen activated protein kinases (MAPK)p₄₂/p₄₄ ^(MARK) mediate protein S membrane to nuclear signaling thatmight be involved in cell proliferation in vascular smooth muscle cells(20). Similar intracellular signaling may mediate the cytoprotectiveand/or neuroprotective effects of protein S.

Bleeding and intracerebral hemorrhage are potential life-hreateningcomplications with anti-thrombotic therapy for stroke includingthrombolytic treatment with tPA (29) or anticoagulant treatment withheparin (27). In addition, tPA is directly toxic for brain cells (30,31)in contrast to cellular neuropro-tection conferred by protein S. Thisstudy indicated that protein S does not adversely affect hemostaticfunction or produce intracerebral hemorrhage, consistent with previousstudies demonstrating that administration of protein S does not causebleeding (28). Thus, protein S and variants thereof serve as prototypesof a new class of agents for clinical stroke with combined systemicanti-thrombotic and anti-inflammatory activities as well as directprotective effects on neurons during cerebral ischemia.

REFERENCES

-   1. Griffin Chapter 113. Regulation of Coagulation. In: William's    Hematology 6^(th) Edition pp. 1435-1449 (2000).-   2. Heeb et al. Binding of protein S to factor Va associated with    inhibition of prothrombinase that is independent of activated    protein C. J. Biol. Chem. 268:2872-2877 (1993).-   3. Hackeng et al. Human protein S inhibits prothrombinase complex    activity on endothelial cells and platelets via direct interactions    with factors Va and Xa. J Biol Chem 269:21051-21058 (1994).-   4. Heeb et al. Protein S binds to and inhibits factor Xa. Proc.    Natl. Acad. Sci. USA 91:2728-2732 (1994).-   5. Koedam et al. Inactivation of human factor VIII by activated    protein C. Cofactor activity of protein S and protective effect of    von Willebrand factor. J. Clin. Invest. 82:1236-1243 (1988).-   6. Rosing et al. Effects of protein S and factor Xa on peptide bond    cleavages during inactivation of factor Va and factor VaR506Q by    activated protein C. J. Biol. Chem. 270:27852-27858 (1995).-   7. Lu et al. Comparison of activated protein C/protein S-mediated    inactivation of human factor VIII and factor V. Blood 87:4708-4717    (1996).-   8. Mahasandena et al. Clinical and laboratory observations:    homozygous protein S deficiency in an infant with purpora    fulmainans. J. Pediatrics 117:750-753 (1990).-   9. Pegelow et al. Severe protein S deficiency in a newborn.    Pediatrics 89:674-676(1992).-   10. Schwarz et al. Plasma protein S deficiency in familial    thrombotic disease. Blood 64:1297-1300 (1984).-   11. Comp et al. Familial protein S deficiency is associated with    recurrent thrombosis. J. Clin. Invest. 74:2082-2088 (1984).-   12. Comp & Esmon. Recurrent venous thromboembolism in patients with    a partial deficiency of protein S. New Engl. J. Med. 311:1525-1528    (1984).-   13. Gladson et al. The frequency of type 1 heterozygous protein S    and protein C deficiency in 141 unrelated young patients with venous    thrombosis. Thromb. Haemost. 59:18-22 (1988).-   14. Green et al. Protein S deficiency in middle aged women with    stroke. Neurology 42:1029-1033 (1992).-   15. Koller et al. Deficiency of both protein C and protein S in a    family with ischemic strokes in young adults. Neurology 44:1238-1240    (1994).-   16. Prats et al. Superior sagital sinus thrombosis in a child with    protein S deficiency. Neurology 42:2303-2305 (1992).-   17. Bostroem et al. Thrombophlébite cérébrale, phlébite surale,    embolies pulmonaires et déficits en protéine S. Rev. Neurol. (Paris)    152:755-758 (1996).-   18. Gasic et al. Coagulation factors X, Xa and protein S, as potent    mitogens of cultured aortic smooth muscle cells. Proc. Natl. Acad.    Sci. USA 89:2317-2324 (1992).-   19. Benzakour et al. Evidence for a protein S receptor(s) on human    vascular smooth muscle cells. Analysis of the binding    characteristics and mitogenic properties of protein S on human    vascular smooth muscle cells. Biochem. J. 308:481-485.(1995).-   20. Kanthou & Benzakour. Cellular effects and signaling pathways    activated by the anti-coagulant factor, protein S, in vascular    cells. Protein S cellular effects. In: Angiogenesis: From the    Molecular to Integrative Pharmacology pp. 155-166 (2000).-   21. Schneider et al. Genes specifically expressed at growth arrest    of mammalian cells. Cell 54:787-793 (1988).-   22. Bellosta et al. Signaling through the ARK tyrosine kinase    receptor protects from apoptosis in the absence of growth    stimulation. Oncogene 15:2387-2397 (1997).-   23. Goruppi et al. Gas6, the ligand of Axl tyrosine kinase receptor,    has mitogenic and survival activities for serum starved NIH-3T3    fibroblasts. Oncogene 12:471-480 (1996).-   24. Nakano et al. Prevention of growth arrest-induced cell death of    vascular smooth muscle cells by a product of growth arrest-specific    gene, gas6. FEBS Lett. 387:78-80 (1996).-   25. Stitt et al. The anticoagulation factor protein S and its    relative, gas6, are ligands for the tyro 3/axl family of receptor    tyrosine kinases. Cell 80:661-670 (1995).-   26. Godowski et al. Reevaluation of the roles of protein S and gas6    as ligands for the receptor tyrosine kinase rse/tyro 3. Cell    82:355-358 (1995).-   27. Zivin. Factors determining the therapeutic window for stroke.    Neurology 50:599-603 (1998).-   28. Arnljots & Dahlback. Antithrombotic effects of activated protein    C and protein S in a rabbit model of microarterial thrombosis.    Arterioscler. Thromb. Vasc. Biol. 15:937-941 (1995).-   29. del Zoppo et al. Hemorrhagic transformation following tissue    plasminogen activator in experimental cerebral infarction. Stroke    21:596-601 (1990).-   30. Wang et al. Tissue plasminogen activator (tPA) increases    neuronal damage after focal cerebral ischemia in wild-type and    tPA-deficient mice. Nature Med. 2:228-231 (1998).-   31. Flavin et al. Microglial tissue plasminogen activator (tPA)    triggers neuronal apoptosis in vitro. Glia 29:347-354 (2000).-   32. Goodnight & Griffin. Chapter 127. Hereditary Thrombophilia. In:    William's Hematology 6^(th) Edition pp. 1697-1714 (2000).-   33. Shibata et al. Anti-inflammatory, antithrombotic, and    neuroprotective effects of activated protein C in a murine model of    focal ischemic stroke. Circulation 103:1799-805 (2001).-   34. Tabrizi et al. Tissue plasminogen activator (tPA) deficiency    exacerbates cerebrovascular fibrin deposition and brain injury in a    murine stroke model. Arterioscler. Thromb. Vasc. Biol. 19:2801-2806    (1999).-   35. Xiang et al. Evidence for p53-mediated modulation of neuronal    viability. J. Neuro. Sci. 16:6753-6756 (1996).-   36. Koretz et al. Pre- and post-synaptic modulators of excitatory    neurotransmission: comparative effects of hypoxia/hypoglycemia in    cortical cultures. Brain Res. 643:334-337 (1994).-   37. del Zoppo. Microvascular response to cerebral    ischemia/inflammation. Ann. NY Acad. Sci. 823:132-147 (1997).

Patents, patent applications, books, and other publications cited hereinare incorporated by reference in their entirety.

All modifications and substitutions that come within the meaning of theclaims and the range of their legal equivalents are to be embracedwithin their scope. A claim using the transition “comprising” allows theinclusion of other elements to be within the scope of the claim; theinvention is also described by such claims using the transitional phrase“consisting essentially of” (i.e., allowing the inclusion of otherelements to be within the scope of the claim if they do not materiallyaffect operation of the invention) and the transition “consisting”(i.e., allowing only the elements listed in the claim other thanimpurities or inconsequential activities which are ordinarily associatedwith the invention) instead of the “comprising” term. Any of thesetransitions can be used to claim the invention.

It should be understood that an element described in this specificationshould not be construed as a limitation of the claimed invention unlessit is explicitly recited in the claims. For example, functional variantsof protein S are known as homologs, mutations, and polymorphisms in thehuman nucleotide and amino acid sequences. In addition, Gas6 analogsand/or receptor agonists (e.g., ligands) of annexin II or members of theTyro3/Axl family may be used as functional equivalents of protein S andits functional variants. Thus, the granted claims are the basis fordetermining the scope of legal protection instead of a limitation fromthe specification which is read into the claims. In contradistinction,the prior art is explicitly excluded from the invention to the extent ofspecific embodiments that would anticipate the claimed invention ordestroy novelty.

Moreover, no particular relationship between or among limitations of aclaim is intended unless such relationship is explicitly recited in theclaim (e.g., the arrangement of components in a product claim or orderof steps in a method claim is not a limitation of the claim unlessexplicitly stated to be so). All possible combinations and permutationsof individual elements disclosed herein are considered to be aspects ofthe invention. Similarly, generalizations of the invention's descriptionare considered to be part of the invention. For example, neuroprotectionmay be manifested by inhibition of apoptosis, promotion of cellsurvival, prevention of neuronal injury and/or cell death, and othergeneral cytoprotective effects.

From the foregoing, it would be apparent to a person of skill in thisart that the invention can be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments should be considered only as illustrative, not restrictive,because the scope of the legal protection provided for the inventionwill be indicated by the appended claims rather than by thisspecification.

1. A method of protecting one or more cell types of a human subject'snervous system, comprising administering to a human subject an amount ofhuman protein S effective to provide neuroprotection, wherein no proteinC or activated protein C is administered, and wherein the human subjecthas a brain injury caused by cerebral ischemia, cerebral hypoxia,cerebral re-oxygenation or any combination thereof.
 2. The method ofclaim 1, wherein there is no deficiency of protein S activity in thehuman subject.
 3. The method of claim 1, wherein the protein Spolypeptide is administered before and/or after diagnosis of disease oranother pathological condition.
 4. The method of claim 1, whereincerebral blood flow in the human subject's brain is increased byadministration of the protein S polypeptide.
 5. The method of claim 1,wherein volume of the human subject's brain which is affected by injury,infarction, edema, or a combination thereof is decreased byadministration of the protein S polypeptide.
 6. A method of treatingstroke in a human subject, comprising administering to a human subjectan amount of human protein S effective to treat stroke, wherein noprotein C or activated protein C is administered, and wherein the humansubject has experienced stroke.
 7. The method of claim 6, wherein thereis no deficiency of protein S activity in the human subject.
 8. Themethod of claim 6, wherein cerebral blood flow in the human subject'sbrain is increased by administration of the protein S polypeptide. 9.The method of claim 6, wherein volume of the human subject's brain whichis affected by stroke is decreased by administration of the protein Spolypeptide.